Method for Highly Multiplexed Quantitation of Peptides by Mass Spectrometry and Labeling Reagent Sets Therefor

ABSTRACT

Disclosed herein are isobaric labeling reagent sets useful for multiplexed quantitation of peptides. The isobaric labeling reagent sets include a collection of at least two isobaric labeling reagents having first and second reporter groups with the same nominal mass but different isotopic substitutions and consequently different exact masses. Mass spectrometric analysis of the labeled samples is performed using a mass analyzer, such as an Orbitrap mass analyzer, capable of adequately resolving the ions of the first and second reporter groups. Reagent sets of the foregoing description may provide a degree of multiplexing in reporter ion quantitation experiments that is expanded relative to conventional labeling reagent sets, thereby reducing the number of chromatographic runs required for analysis and improving sample throughput.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation under 35 U.S.C. §120 and claims thepriority benefit of co-pending U.S. patent application Ser. No.14/574,854 filed Dec. 18, 2014, which is a divisional under 35 U.S.C.§121 of U.S. patent application Ser. No. 14/000,856, filed Aug. 21,2013, now U.S. Pat. No. 8,940,546, which is the United States NationalStage Application, under 35 U.S.C. 371, of International ApplicationPCT/US2013/040402 having an international filing date of May 9, 2013,which claims the priority benefit of U.S. Provisional Patent ApplicationSer. No. 61/645,311 for “Method for Highly Multiplexed Quantitation ofPeptides by mass Spectrometry and Mass Labels Therefor”, filed May 10,2012. The disclosures of each of the foregoing applications areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to methods for multiplexedquantitation of peptides by mass spectrometry utilizing isobaric masslabels, and more particularly to a set of isobaric mass labels usefulfor providing enhanced sample multiplexing capabilities and methodsemploying such sets of mass labels.

BACKGROUND OF THE INVENTION

Isobaric mass tagging or labeling is a method for determining therelative abundance of a given peptide across multiple samples by massspectrometry. Each sample, typically representing a distinct biologicalcondition, is reacted with a different isotopic variant of a labelingreagent. The samples are mixed together and analyzed by MS/MS. When agiven peptide (isobaric molecules collected from all conditions) isisolated and fragmented, reporter ions arising from the various labelingreagents have different nominal masses, identifying the origin of eachpeptide, and allowing relative quantification of the peptide acrosssamples. Sets of isobaric mass tagging reagents are commerciallyavailable from Thermo Fisher Scientific Inc, under the trade name TandemMass Tags (TMT), and from AB Sciex under the trade name Isobaric Tagsfor Relative and Absolute Quantitation (iTRAQ). The structure andutilization of isobaric mass tags has been described extensively in thepatent prior art (see, for example, U.S. Pat. No. 7,816,304 by Schmidtet al. and U.S. Pat. No. 7,732,378 by Thompson et al., the disclosuresof which are both incorporated herein by reference) as well as in thescientific literature (see, for example, Ross et al., “MultiplexedProtein Quantitation in Saccharomyces cerevisiaie Using Amine-reactiveIsobaric Tagging Reagents”, Molecular & Cellular Proteomics, 3(12), pp.1154-1167 (2004); and, Dayon et al., “Relative Quantification ofProteins in Human Cerebrospinal Fluids by MS/MS Using 6-Plex IsobaricTags”, Analytical Chemistry, Vol. 80, pp. 2921-2931 (2008)).

Considerable effort has been invested in increasing the “multiplex”capacity of the isobaric mass tag system. For example, the “sixplex TMT”reagent set has additional value over the “fourplex iTRAQ” reagent setbecause it allows the user to compare more samples with fewerchromatographic runs. The multiplex capacity of the system is ultimatelylimited by the number of atoms in the reporter region of the isobaricreagent available for isotopic substitution and the difficulty and costassociated with introducing a large number of isotopic substitutions.

U.S. Patent Application Publication No. 2010/0029495 to Schaeferdescribes an approach to expanding multiplexing capacity by providing aset of labeling reagents which includes a plurality of reagents havingreporter groups of the same mass but of different molecular structures.Its technique involves performing a second stage of fragmentation (MS³)to cause fragmentation of the reporter ions, such that each same-massreporter group yields characteristic product ions that may bedistinguished in the mass spectrum, thereby permitting assignment of therelevant intensity to the corresponding labeled sample. While thisapproach may provide for a greater degree of multiplexing, therequirement of performing MS³ to distinguish different reporter ions ofthe same mass complicates the analysis, and may reduce sensitivity andthroughput.

SUMMARY

In accordance with embodiments of the present invention, a set ofisobaric labeling reagents are provided wherein at least two of thelabeling reagents have reporter groups with the same elementalcomposition and nominal (integer) mass but different isotopicsubstitutions and consequently different exact masses; for example, onereporter group may have one or more carbons substituted with the ¹³Cisotope, while the other reporter group may have a corresponding numberof nitrogens substituted with the ¹⁵N isotope (or a combination of ¹³Cand ¹⁵N isotopes). The reporter ions produced by ionization andfragmentation of peptides labeled with these at least two reagents(i.e., during MS/MS analysis) have exact masses that are sufficientlydifferent to enable them to be resolved from one another in a massspectrum acquired using an Orbitrap, Fourier Transform/Ion CyclotronResonance (FT/ICR) or other instrument capable of high-resolution,accurate mass operation. The inclusion of two or more labeling reagentshaving reporter groups with the same nominal mass but different isotopicsubstitutions can significantly increase the multiplexing capability ofa labeling reagent having a specified reporter group structure, therebyenabling a greater number of samples to be analyzed within a singlechromatographic run and avoiding the uncertainties arising fromrun-to-run variations, which compromise the ability to directly comparedata acquired for different samples. The increased multiplexingcapability enabled by embodiments of the present invention also presentsthe advantage of increasing overall sample throughput, thus decreasingthe amount of time required to complete an experiment and/or increasingthe numbers of sample that may be analyzed within a given time.

The present invention also provides a method for highly multiplexedMS/MS analysis of a plurality of samples. In certain illustrativeimplementations, the samples may correspond to biological replicates,points in a time course, different cell lines, various perturbations ofthe biological system, etc. The method includes providing a set ofisobaric labeling reagents, wherein at least two of the reagents havereporter groups that possess the same nominal mass and elementalcomposition but have different isotopic substitutions (as used herein,two reporter groups have different isotopic substitutions if onereporter group has a different number of one substituent, e.g., ¹³C or¹⁵N, relative to the other reporter group). Each one of the samples isreacted with a different reagent of the labeling reagent set to yieldlabeled analytes (e.g., molecules comprising a peptide of interest boundto a labeling reagent). The samples are then combined and subjected toMS/MS analysis, whereby the labeled analytes are (preferably) isolatedand fragmented under controlled conditions, for example by collisionallyactivated dissociation (CAD). Product ions produced by dissociation ofthe labeled analytes include reporter ions, each having a characteristicmass. The mass analyzer is operated to acquire an MS/MS mass spectrum ata sufficiently high resolution to enable reporter ions of the samenominal mass but differing isotopic substitutions to be resolved asseparate peaks. The relative quantities of the analyte(s) of interestmay then be determined from the reporter ion intensities in the MS/MSspectrum, whereby each reporter ion peak corresponds to a differentsample.

BRIEF DESCRIPTION OF THE FIGURES

In the accompanying drawings:

FIG. 1 is a symbolic diagram depicting the component groups of a genericisobaric labeling reagent;

FIG. 2 depicts the isotopic variants of the members of the sixplex-TMTreagent set; and

FIG. 3 is a diagram depicting a method for determining the relativeabundance of a given peptide across multiple samples by massspectrometry, using a isobaric labeling reagent set composed accordingto an embodiment of the invention.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention is described below in reference to certain specificembodiments. It should be recognized that the embodiments describedbelow are presented by way of illustration, and the invention should notbe construed as being limited to the disclosed embodiments, but insteadshould be accorded the full scope of the claims presented below.

The structure and synthesis of isobaric labeling reagents are well-knownin the art and need not be discussed in detail herein. Typically, anisobaric labeling reagent is formed from three linked groups ormoieties, as depicted in FIG. 1, consisting of a reporter group, a massnormalizing group, and a reactive group. Each labeling reagent has areporter group mass that is different from that of the other labelingreagents of the set (in conventional labeling reagent sets, the reportergroups have different nominal masses); the isobaric character of thelabeling reagents is effected by providing a mass normalizing grouphaving a mass that “balances out” the differential mass of the reportergroup, such that all composite nominal masses are the same. Theadjustment of reporter group and mass normalizing group masses isachieved by isotopic substitution, as described below. The reactivegroup reacts with the free amino-terminus of peptides to form a labeledanalyte, which is ionized in a mass spectrometer, typically by theelectrospray ionization technique. The reporter group is attached to themass normalizing group by a cleavable linker, which fragments under CADor equivalent conditions to release the reporter group, which retainscharge and is thus observable in the product ion spectrum.

The current commercially available sixplex TMT system (referred to as“TMT-6” and sold by the Pierce Protein Biology Products business ofThermo Fisher Scientific Inc.) gives rise to six isotopic variants ofthe same reporter group C₈H₁₆N, as shown in FIG. 2. The six variantscorrespond to reporter groups with 0, 1, 2, 3, 4, and 5 additionalneutrons, corresponding to nominal masses 126, 127, 128, 129, 130, and131 respectively. When an extra neutron is placed on a carbon atom(¹²C→¹³C), the increase in mass (1.00335 Dalton(Da)) is slightlydifferent than when it is placed on a nitrogen atom (¹⁴N→¹⁵N, 0.99703Da). For example, the group with nominal mass 127 shown above is a ¹³Csubstituted variant. Alternatively, a reporter group of nominal mass 127could be formed by substituting ¹⁵N instead of ¹³C. The ¹³C form is 6.32milliDalton (mDa) heavier than the ¹⁵N form.

Given a mass spectrometry system that cannot accurately quantify a pairof ions closer than one mass unit apart, the set of isobaric labelingreagents will incorporate either the ¹³C or ¹⁵N substituted form of thereporter ion, but not both together. However, if a mass spectrometer isemployed that is capable of accurately quantifying two species that havean exact mass difference of 6.32 mDa, it is possible to use bothreporter ions (the ¹³C and ¹⁵N substituted forms) to encode sampleinformation. Quantifying the reporter ions given in this examplerequires that the analyzer's “quantitative resolving power” exceed20,000 (20K). We may define quantitative resolving power as m/dm, wheredm is the smallest mass separation for which distinct ion species withmasses m and m+dm can be accurately quantified. We observe that mostcommercial instruments require baseline separation of adjacent ions foraccurate quantification. This means that the quantitative resolvingpower may be a factor of 2-5 times less than the resolutionspecification. Commercially available FTMS analyzers including theOrbitrap mass spectrometer and FT-ICR MS instruments are capable of farexceeding 100K in resolution, so that the quantitative resolving powerclearly exceeds 20K. Certain time-of-flight (TOF) instruments also haveresolution in excess of 20K, but it is unclear whether they havesufficient quantitative resolving power for the proposed application.

The quantitative resolving power of an Orbitrap or other FourierTransform analyzer is known to be roughly proportional to the transientduration over which the mass spectrum is acquired, which is referred toherein as the scan time. Otherwise expressed, the scan time required toaccurately quantify adjacent reporter ion peaks is inverselyproportional to the exact mass difference between the adjacent reporterion peaks (it is noted that in the context of discussion of massspectrometric analysis, the term “mass” and its variants are used hereinas shorthand for the measured mass-to charge ratio (m/z)). The inventorshave demonstrated the ability to accurately quantify peaks having a 6.32mDa exact mass difference in a commercially available Orbitrapinstrument operated with a scan time of less than 32 milliseconds (ms).In this example, very high MS/MS scan rates, up to 30 Hz, would bepossible, without losing the ability to resolve and accurate quantifythe relative abundances of the reporter ions. As will be discussedbelow, accurate quantification of more closely spaced reporter ion peaksis feasible, but necessitates longer scan times and consequently lowerscan rates. While commercially available versions of the Orbitrapanalyzer are adequate to provide the required resolving power atacceptable scan rates, scan rates and throughput may be furtherincreased by the use of data acquisition and processing techniques suchas matched-filter linear deconvolution.

In accordance with embodiments of the present invention, themultiplexing capability of a isobaric reagent set having a givenreporter group structure may be expanded by inclusion of reporter groupshaving the same elemental composition and nominal mass, but differentnumbers of ¹⁵N and ¹³C substitutions (and consequently different exactmasses), and may be expanded still further by the inclusion of nominallyisobaric reporter groups having different numbers of ¹⁵N, ¹³C and ²Hsubstitutions. The table set forth below lists a total of 36 candidatereporter groups, each having the molecular structure utilized in thecommercially available TMT-6 labeling reagents and having (except forthe reporter ion having a nominal mass of 126) one or more isotopicsubstitutions. The table indicates, for each reporter group, the nominaland exact masses, the number of ¹⁵N, ¹³C and ²H substitutions, and thedifferential mass (designated as Δmass) representing the exact massdifference relative to the lower-mass adjacent reporter group within acollection of reporter groups having the same nominal mass (i.e., thereporter group appearing immediately above in the table). The reportergroups currently used for the TMT-6 set are marked as TMT-6 in the notescolumn. As indicated in the table, it is possible to select a set of 11reporter groups, comprising the nominal mass 126 reporter group, plus acollection of two reporter groups each at nominal masses 127-131, witheach of the reporter ions at nominal masses 127-131 representing zero orone ¹⁵N substitution combined with one or more ¹³C substitutions, witheach reporter ion in the set being separated from adjacent reporter ionswithin the set by at least 6.3 mDa. As noted above, it is possible toquantify peaks having an accurate mass difference of 6.3 mDa with a massanalyzer having a nominal resolving power of 20K. The five additional(relative to the TMT-6 set) reporter groups are marked as “TMT-11” inthe notes column of the table.

²H substitutions can also be used to form a much larger set of reportergroups. Adding a neutron to ¹H to form ²H increases its mass by 1.00628Da. In principle, we could form (8+1)*(16+1)*(1+1)=306 differentisotopic forms of C₈H₁₆N, representing all possible substitutionpatterns. However, if we restrict ourselves to substitutions of nominalmasses 126-131 (analogous to the TMT-6 reagent set), there are 36different isotopic forms. These 36 forms comprise the candidate reportergroups set forth in the table below.

Out of the total of 36 candidate reporter groups, a set comprised ofthirty groups can be chosen so that all are separated by 2.93 mDa orgreater; there are six pairs that separated by just 0.46 mDa. Weselected one representative from each of these pairs for inclusion intoa set of 30 reporter groups. The six isotopic forms eliminated fromconsideration are indicated as such in the table.

Nominal Exact Mass Mass No. ¹⁵N No. ¹³C No. ²H Δmass Notes 126 126.128270 0 0 TMT-6 127 127.1253 1 0 0 TMT-11 127 127.13162 0 1 0 0.00632 TMT-6127 127.13455 0 0 1 0.00293 128 128.12865 1 1 0 TMT-11 128 128.13158 1 01 0.00293 128 128.13497 0 2 0 0.00339 TMT-6 128 128.1379 0 1 1 0.00293128 128.14083 0 0 2 0.00293 129 129.132 1 2 0 TMT-11 129 129.13493 1 1 10.00293 129 129.13786 1 0 2 0.00293 Eliminated 129 129.13832 0 3 00.00046 TMT-6 129 129.14125 0 2 1 0.00293 129 129.14418 0 1 2 0.00293129 129.14711 0 0 3 0.00293 130 130.13535 1 3 0 TMT-11 130 130.13828 1 21 0.00293 130 130.14121 1 1 2 0.00293 Eliminated 130 130.14167 0 4 00.00046 TMT-6 130 130.14414 1 0 3 0.00247 Eliminated 130 130.1446 0 3 10.00046 130 130.14753 0 2 2 0.00293 130 130.15046 0 1 3 0.00293 130130.15339 0 0 4 0.00293 131 131.1387 1 4 0 TMT-6 131 131.14163 1 3 10.00293 131 131.14456 1 2 2 0.00293 Eliminated 131 131.14502 0 5 00.00046 TMT-11 131 131.14749 1 1 3 0.00247 Eliminated 131 131.14795 0 41 0.00046 131 131.15042 1 0 4 0.00247 Eliminated 131 131.15088 0 3 20.00046 131 131.15381 0 2 3 0.00293 131 131.15674 0 1 4 0.00293 131131.15967 0 0 5 0.00293

A nominal quantitative resolving power of about 40K would be required toquantify reporter ions having a 2.93 mDa separation; for 0.46 mDa, thequantitative resolving power required is more than six times greater.This quantitative resolving power may be achievable in the Orbitrapanalyzer, but the required transient duration (scan time) would be morethan six times longer relative to a spectrum acquired at a resolvingpower of 40K. Doing so would significantly reduce scan rate (i.e., thenumber of mass spectra acquired per unit time), and thus operating themass analyzer at the higher resolving power may not be an acceptabletradeoff to increase the multiplex capacity an additional 20% from 30 to36. Peaks corresponding to the thirty remaining reporter ions (those noteliminated due to their mass proximities to adjacent reporter ionswithin a nominal mass group) may be adequately resolved in an Orbitrapmass analyzer operated with a scan time of about 64 ms. This iscompatible with MS/MS scan rates up to 15 Hz.

It is possible that certain of the isotopic forms that comprise the30-reporter group set listed in the table may be difficult tosynthesize, and thus may not be realizable in a commercially practicalmanner. However, in any case, the use of labeling reagents having one ormore reporter groups having the same nominal mass but different isotopicsubstitutions, combined with MS/MS analysis by mass spectrometerscapable of sufficiently high resolution to resolve the nominallyisobaric reporter ions as separately quantifiable peaks, makes possiblemuch higher multiplex capacity than would be possible with unitresolution.

It should be understood that the present invention should not beconstrued as being limited to any particular reporter group structure.The reporter group structure discussed above (which is employed for thecommercially available TMT-6 isobaric labeling reagent set, and has amolecular formula of C₈H₁₆N) is provided only by way of an illustrativeexample. The principle of the invention, in which multiplexing capacityis expanded by inclusion of labeling reagents having nominally isobaricreporter groups, may be applied to any number of reporter groupstructures. Examples of other reporter group structures that may beadapted for the present invention include those described in U.S. Pat.Nos. 7,294,456; 7,732,304; 7,816,304; and 7,825,069 as well as U.S.Patent Application Publication Nos. 2010/0178710; 2010/0029495;2010/0167267; and 2011/0111513, the entire disclosures of which areincorporated herein by reference. Further, the newly demonstratedability to accurately quantify isobars at high scan rates could motivatea redesign of the reporter region of the TMT reagent to provide evenmore isotopic combinations to further increase the multiplex capacity.

It should be further recognized that the highly multiplexed labelingreagent sets described herein do not require any special adaptation ormodification to the mass-normalizing and peptide-reactive groups of thelabeling reagents. More particularly, the isotopic substitutions on themass-normalizing groups need only balance out the differences in thenominal masses between or among the reporter groups, rather thancompensate for the differences in exact masses. It is noted that thelabeled analytes may thus exhibit differences in their exact masses, butsuch differences will be sufficiently small so as to not have asubstantial impact on either chromatographic retention times or theco-isolation of labeled analytes for MS/MS analysis.

Labeling reagent sets of the foregoing description may be utilized insubstantially the same manner as commercially available labeling reagentsets, such as TMT-6. As depicted in FIG. 3, the analysis involves aninitial step 310 of separately and identically preparing each one of aplurality of samples via known techniques suitable for purificationand/or derivatization of analyte proteins and/or peptides present in thesamples, which may include immunoaffinity capture/enrichment,denaturing, centrifugation/filtering, and proteolytic digestion. In atypical experiment, each sample may correspond to a different point of atime series with a corresponding one of the labeling reagents in orderto form labeled analytes, e.g., peptides.

Next, each of the prepared samples is mixed with a different one of thelabeling reagent of the isobaric reagent labeling set to produce labeledpeptide analytes, for example via attachment of the labeling reagent tothe amino terminal of peptides present in the sample, step 320. Asdiscussed above, the isobaric reagent set will include a collection ofat least two labeling reagents, in which the two labeling reagents havereporter groups having the same nominal masses but different isotopicsubstitutions; others of the labeling reagents will have reporter groupshaving different nominal masses. The prepared and labeled samples arethen mixed together into a single combined sample, step 330. Off-lineand/or on-line chromatographic separation techniques, for example strongcation exchange chromatography, may be utilized to separate componentsor groups of components from each other to simplify mass spectrometricanalysis, per step 340. The combined sample (or chromatographicfractions thereof) is then subjected to MS/MS analysis employing asuitable mass spectrometer, such as the Thermo Scientific Q Exactive orOrbitrap Elite mass spectrometers. As known in the art, such analysisconsists of first ionizing the sample mixture, step 350, which may beaccomplished, for example, by electrospray ionization. The resultantions are then mass selected (using, for example, a quadrupole massfilter or ion trap) to isolate one or more precursor ions of interest(i.e., those corresponding to a labeled peptide analyte), andfragmenting the precursor ions by CAD or other technique to generateproduct ions, step 360, which will include the reporter ions produced byfragmentation of the labeled analytes. In step 370, the abundances ofreporter ions at each characteristic value of mass-to-charge ratio aredetermined by acquisition of a mass spectrum of the product ionsproduced in step 360. As discussed above, the product ion mass spectrumis acquired using a mass analyzer capable of and operated at a resolvingpower high enough to resolve the nominally isobaric reporter ions ofdiffering isotopic composition. The mass analyzer may be, for example,an Orbitrap or FT/ICR analyzer. Per the foregoing example, aquantitative resolving power of 20K is adequate to resolve all reporterions in the 11 reagent set identified in the table above (having aminimum separation between adjacent reporter ions of 6.32 mDa), and aresolving power of 40K is adequate to resolve all ions in the 30 reagentset (having a minimum separation of 2.93 mDa). Each reporter ion peak inthe spectrum is representative of and assignable to a different sample,and the relative quantities of an analyte of interest present in thesamples may be determined from the relative intensities of the reporterion peaks appearing at each characteristic m/z value, in the mannerdescribed in the art.

Various data processing methods known in the art may be utilized toincrease the signal-to-noise ratio of the measured peaks and therebyimprove the accuracy of the relative quantitation. Furthermore, themeasured intensities may be adjusted (in accordance with knownapproaches, such as is described in U.S. Pat. No. 7,105,806 by Pappin etal.) to account for isotopic impurities in the labeling reagents, whichresult in the production of up-mass and down-mass side peaks.

1. An isobaric labeling reagent set for use in quantitative measurementof peptides by mass spectrometry, comprising: a plurality of differentisobaric labeling reagents, each reagent comprising a reporter group, amass normalizing group, a peptide-reactive group, and a cleavable linkerattaching the reporter group to the mass normalizing group, each one ofthe plurality of isobaric labeling reagents having the same nominalmass; each one of the isobaric labeling reagents having a reporter groupwith a mass different from the masses of the reporter groups of each ofthe other isobaric labeling reagents, each reporter group having thesame elemental composition; wherein a first collection of at least twoof the isobaric labeling reagents have reporter groups of the samenominal mass but different exact mass by having different numbers of atleast one of ¹³C, ¹⁵N and ²H substitutions; and wherein the reportergroups of each of the isobaric labeling reagents have a structureconsisting of an N-methyl pyridyl group or a group selected from thefollowing groups: —NH₂, —NR₂, —NR₃ ⁺, —SR₃ ⁺, —CO₂—,

wherein R is hydrogen or is a substituted or unsubstituted aliphatic,aromatic, cyclic or heterocyclic group.
 2. The isobaric labeling reagentset of claim 1, wherein the first collection includes isobaric labelingreagents having reporter groups with different numbers of ¹³Csubstitutions.
 3. The isobaric labeling reagent set of claim 1, whereinthe first collection includes isobaric labeling reagents having reportergroups with different numbers of ¹⁵N substitutions.
 4. The isobariclabeling reagent set of claim 1, wherein the first collection includesisobaric labeling reagents having reporter groups with different numbersof ²H substitutions.
 5. The isobaric labeling reagent set of claim 1,wherein the masses of reporter groups of the isobaric labeling reagentsin the first collection differ from each other by at least 2.93 mDa. 6.The isobaric labeling reagent set of claim 1, wherein a secondcollection of at least two of the isobaric labeling reagents havereporter groups of the same nominal mass but different exact mass byhaving different numbers of at least one of ¹³C, ¹⁵N and ²Hsubstitutions, the nominal mass of the reporter groups of the secondcollection being different from the nominal mass of the reporter groupsof the first collection.